Most cell structures in the human and animal body are organised in three-dimensions. This leads to complex intercellular interactions that cannot be mimicked in two-dimensional monolayers of cell cultures (Pampaloni, Ansari and Stelzer, 2013). However, two-dimensional cell culture has been the paradigm for typical in vitro cell culture. It has been demonstrated that cells behave more natively when cultured in three-dimensional environments but the formation of three-dimensional cell culture is difficult, expensive and laborious to generate.
Cellular spheroids are an example of a three-dimensional cell culture model and was among the first to be discovered and applied in basic research and clinical pharmacology. Cellular spheroids are aggregated cell clusters with a typical diameter of hundreds of microns. There are many techniques currently available for spheroid formation, such as the hanging drop method or rotational stirrers. Typically, culturing a cellular spheroid can take up to four days and has a 50% success rate.
Numerous studies have reported the printing of cells using drop-on-demand type technology. Faulkner-Jones et al. (2013) printed cells suspended in medium using the drop-on-demand method with a cell density of approximately 1×105 cells/ml in DMEM. Ferris et al. (2015) reported the printing of 2×106 cells/ml in bio-ink (gellan gum) using a commercially available inkjet printhead. Xu et al. (2014) reported the printing of cells in bio-ink at a density of 1×107 cells/ml. It was also reported that the most widely used cell concentrations in cell printing are between 105 and 107 cells/ml. While many previous studies describe printing of cells using drop-on-demand technology, there are no reports of printing a high cell density (greater than 107 cells/ml) in bio-ink via the drop on demand method.
In order to form a cellular spheroid, for example, a high density of cells (5×107−2×108 cells/ml) must be aggregated into a small spherical cell cluster. The hanging drop technique achieves this by suspending droplets of culture medium containing isolated cells and allowing the cells to aggregate over three to seven days. Generating cellular spheroids using a drop-on-demand device presents difficulties due to the high viscosity associated with high cell densities and the small feature size in a drop-on-demand device. For this reason, in previous attempts to print tissue structures using spheroids, the cellular spheroids were firstly formed manually, loaded into a 3D printer and then printed to form a tissue structure. Tan et al. (2014) reported the 3D printing of preformed cellular spheroids via the extrusion method. Tan et al used a pressurised Pasteur pipette system as the droplet dispensing nozzle, which resulted in a continuous deposition of preformed spheroids. Marga et al. (2012) also reported the 3D printing of preformed spheroids via the extrusion method. In contrast, capillary micropipettes were used to contiguously arrange the cellular aggregates in a single line. Similarly, Mironov et al. (2009) also reported the 3D printing of preformed cellular spheroids via the extrusion method.
The present inventors have developed a process for preparing a 3D tissue culture model suitable for in vitro cell culture assays.